Fibrinolysin-desoxyribonuclease for enzymatic debridement



United States Patent 2 Claims. (Cl. 167-73) This application is a continuation of our co-pending application, Serial No. 9,171, filed February 17, 1960, now abandoned.

This invention relates to improved enzymatic compositions, and more particularly, to enzymatic compositions comprising fibrinolysin and desoxyribonuclease.

Various proteolytic enzyme preparations including trypsin, papain, and fibrinolysin are known to be of value in the removal of necrotic elements from ulcerating Wounds and lesions of various sorts. Such use of proteolytic enzymes for the removal of necrotic debris in lesions has been established as a means of promoting healing and as an adjunct in surgical procedures such as skin grafting. However, the use for these purposes of enzymatic compositions known in the prior art has been restricted by their limited ability to cause chemical dissolution of the wide variety of macromolecular debris commonly encountered in infected and poorly healing lesions.

It is an object of the present invention to provide enzymatic compositions of greater efiicacy in enzymatic debridement. The term enzymatic debridement is defined as the removal by enzymatic means, of protein and non-protein macromolecular debris within the body or on body surfaces; such macromolecular debris can include fibrin, eschars, blood clots, clotted elements of hematomas, purulent exudates, sloughs from infected wounds, and debris of like character.

An object of the present invention is to provide improved compositions broadly effective in enzymatic debridement against a variety of substrates.

Another object of this invention is to provide improved compositions for enzymatic debridement which are relatively free of the side reactions associated with enzymatic compositions of bacterial origin, and whose activity does not depend on the presence of enzyme precursors extrinsic to the compositions themselves.

A further object of this invention is to provide improved compositions for enzymatic debridement having enhanced utility by virtue of antibacterial properties,

Still another object of this invention is to provide methods for enzymatic debridement by the administration of such improved compositions.

These as well as other objects which will appear hereinafter are achieved by providing novel compositions comprising fibrinolysin and desoxyribonuclease, optionally in combination with an antibacterial antibiotic. The fibrinolysin and desoxyribonuclease employed in these compositions are of mammalian origin and can be obtained from such sources as cattle and humans. For example, the fibrinolysin can be derived from bovine plasma-and the desoxyribonuclease from bovine pancreas.

The compositions of the invention are obtained by mixing fibrinolysin and desoxyribonuclease and providing the mixture in a pharmaceutically-acceptable form. The mixture is constituted to have a selected ratio of fibrinolysin and desoxyribonuclease. Fibrinolysin is a proteolytic and more particularly, a fibrinolytic enzyme. The potency of a fibrinolysin preparation is measured and expressed in terms of its ability to lyse a standard fibrin clot. The method of assay is based on the work of Loomis et 211., Archives of Biochemistry, 12, l (1947), and

dissolve 1 ml. of 0.3%

3,208,908 Patented Sept. 28, 1965 is described more fully hereinafter. A unit of fibrinolysin is defined as that amount of fibrinolysin which will bovine fibrin clot in 120 seconds at pH 7.2 and 45 C. in an isotonic saline system buffered with imidazole. Desoxyribonuclease is a nucleolytic enzyme capable of attacking desoxyribonucleoprotein and desoxyribonucleic acid. The potency of a desoxyribonuclease preparation is measured and expressed in terms of its ability to reduce the relative viscosity of a solution of highly polymerized desoxyribonucleic acid, sodium salt. The method of assay is based on the work of Christensen, Journal of Clinical Investigation 28, 163 (1949), and is described more fully hereinafter. A unit of desoxyribonuclease is defined as that amount of desoxyribonuclease which causes a fall in relative viscosity of 1.0 under the standard assay conditions, with the restriction that this fall in relative viscosity of 1.0 be between relative viscosity: 4.0 and relative viscosity=3.0 during a 10 minute period. The compositions of the invention are preferably formulated to have a ratio of 1 unit of fibrinolysin to 20-5000 units of desoxyribonuclease, and the most usual ratio is 1 unit of fibrinolysin to 500-4000 units of desoxyribonuclease.

A composition suitable for use directly or for formulation into any of a variety of forms for parenteral and topical administration is obtained by mixing fibrinolysin and desoxyribonuclease in cold dilute saline in sterile, pyrogen-free equipment, adding selected additional constituents, optionally including a solubilizer such as sucrose, glucose, sodium lauryl sulfonate, dextran or polyvinylpyrrolidone, and a preservative, filtering the solution and lyophilizing the filtrate. The resulting powder can be formulated into a dusting powder for direct application to Wounds. It can also be redissolved in dilute saline or other pharmaceutically-acceptable diluents for parenteral or topical administration, or it can be formulated into a variety of other pharmaceutical forms such as ointments, tablets, films, and the like. To increase stability, the composition is preferably maintained in relatively anhydrous form until just prior to use. In order to provide for the possibility of some loss of potency upon storage as well as variability in the assay procedure, it is customary to measure up to a 40% excess of fibrinolysin and up to a excess of desoxyribonuclease in preparing fresh formulations. It is considered noteworthy that neither the fibrinolysin nor the desoxyribonuclease has a deleterious effect on the activity of the other when both are present in the composition.

Compositions of fibrinolysin and desoxyribonuclease which are particularly advantageous for use in many applications are those which contain an antibacterial antibiotic as an additional therapeutic ingredient. Chloramphenicol, streptomycin, dihydrostreptomycin, penicillin and tyrothricin are representative of the wide variety of antibiotics which can be used in these compositions without adverse effect on enzymatic activity.

Pharmaceutically-acceptable solid or liquid diluents can be incorporated into these compositions. Solid carriers and diluents suitable for use include sugars such as lactose and sucrose; cellulose derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, methyl cellulose, and cellulose acetate phthalate; cornstarch; stearic acid and magnesium stearate. Non-aqueous liquid carriers and diluents can be used for compositions intended to be stored whereas aqueous diluents can be used when the material is ready for use. Ointments are preferably made up with a petroleum base or other non-aqueous media. Additives are included as required to increase solubility and stability.

The compositions of the invention are effective for enzymatic debridement when administered parenterally or topically. They promote the dissolution of undesirable exudates on body surfaces and mucous membranes, and facilitate healing. The compositions are useful in treating superficial ulcers, burns, and infections, in cleaning abscess cavities, in removing necrotic debris associated with vaginitis and cervicitis, in promoting the healing of ulcerated lesions and wound infections, and generally to produce enzymatic debridement of fibrinous and purulent material. The results obtained with these novel compositions are superior to those obtained when fibrinolysin and desoxyribonuclease are used separately, reflecting the fact that the combination causes a concerted attack on the macromolecular debris in the area undergoing treatment without the persistence of a resistant pyogenic or fibrin barrier. The novel compositions containing fibrinolysin, desoxyribonuclease and an antibacterial antibotic such as chloramphenicol are especially superior to the same components employed individually. The enzyme combination potentiates the. antibacterial effect of the antibiotic, and such compositions are especially effective in the topical treatment of infected burns and other lesions where infection is shielded and nourished by macromolecular debris.

The invention is illustrated by the following examples:

EXAMPLE 1 Preparation of lyophilized powder containing fibrinolysin and desoxyribonuclease A solution in dilute saline of fibrinolysin (from bovine plasma) assaying about 30-150 units per ml. is kept in a deep freeze until the preparation is begun. A quantity of the solution sufficient to provide 3500 units of fibrinolysin is then removed and allowed to thaw within a period of 72 hours or less, in a manner such that the temperature does not exceed 3 012 C. at any time. To this solution' is added 25 ml. of sterile physiological salt solution containing 5 g. of sucrose to maintain stability and increase the solubility of the final product. This dilution and the operations which follow are carried out at 0-5" C. using sterile, pyrogen-free equipment and solutions.

There is then added 3 million units of desoxyribonuclease (from bovine pancreas) and the pH of the solution is adjusted to 7.2 ;0.1 with 0.1 N sodium hydroxide solution. The volume is then adjusted to 150 ml. by the addition of sterile physiological salt solution containing suflicient sodium ethylmercurithiosalicylate to yield 0.1 mg./ 1.5 m1. of the resulting bulk solution. This solution is aseptically filtered through Selas candles of appropriate size and porosities (#10, #01, #015 and #02, respectively). The use of Ertel pads #3 thru #EO has also been found to yield satisfactory results. Sufficient sterile salt solution is then passed through the filtering apparatus to rinse out active material and the washings are added to the main filtrate in sufiicient quantity to yield a total volume of 200 ml. After thorough mixing, the solution is filled in 2.0 ml. portions into sterile ,vials or ampoules which are snap frozen at -25 to -60 C. Following lyophilization of the solution to a powder, the vials or ampoules are capped or sealed. Each vial contains approximately 35 units of fibrinolysin and 30,000 units of desoxyribonuclease. For use, the vial or ampoule is opened and the contents are dissolved in the quantity of saline (typically ml.) required to give the, desired concentration.

EXAMPLE 2 Preparation of ointment containing fibrinolysin and desoxyribonaclease A powdered mixture of 140 units of fibrinolysin and 120,000 units of desoxyribonuclease is prepared and mixed with suflicient petroleum jelly to give a total weight of 100 g. A high viscosity petroleum base such as Jelene 50W can be used as the petroleum jelly. The resulting ointment is passed through an ointment mill and filled into tubes. Each gram of the ointment-contains approximately 1.4 units of fibrinolysin and 1200 units of desoxyribonuclease.

4 EXAMPLE 3 Preparation of ointment containing fibrinolysin, desoxyribonuclease anal chloramphenicol A powdered mixture of 140 units of fibrinolysin and 120,000 units of desoxyribonuclease is mixed with 1.05 g. of chloramphenicol and the mixture dispersed in sufficient petroleum jelly to give a total weight of g. The ointment as thus prepared is passed through an ointment mill and filled into tubes. Each gram of the ointment contains approximately 1.4 units of fibrinolysin, 1200 units of desoxyribonuclease and 10.5 mg. of chloramphenicol.

EXAMPLE 4 Preparation of compressed vaginal tablets containing fibrinolysin and desoxyribonuclease A mixture is prepared by blending 2118 g. of powdered milk sugar, 2265 g. of anhydrous dextrose, 60 g. of choramphenicol and 440 g. of dried, powdered potato starch. An ethyl cellulose solution is prepared by mixing 127 g. of an ethyl cellulose (10 cps.) 20% w./w. solution in alcohol with rapid stirring in 508 g. of alcohol, and to it are added, 8.65 ml. of a 2% aqueous solution of PD. & C. Red No. 2, 8.65 ml. of a 3.2% aqueous solution of PD. & C. Yellow No. 5, and 7.15 ml. of a 0.2% aqueous solution of ED. & C. Blue No. 1. The blended mixture of milk sugar, dextrose, chloramphenicol and potato starch is granulated with the ethyl cellulose solution and the granulation is completed with an additional. 147 ml. of alcohol. The granulated mixture is passed through a 6 mesh stainless steel screen and dried at 1l0120 F. The dried material is passed througha 14 mesh screen mounted on an oscillator and blended with an intimate mixture of 180 g. of dried, powdered potato starch, g. of sifted talc and 240 g. of a mixture of fibrinolysin and desoxyribonuclease (containing 0.2 unit of fibrinolysin and 150 units of desoxyribo-nuclease per mg.) which has been passed through a 60 mesh screen. Magnesium stearate (9 g.) is then blended in and the mixture is compressed to yield about 6,000 tablets suitable for vaginal administration, each containing approximately 8 units of fibrinolysin, 6,000 units of desoxyribonuclease, and 10 mg. of chloramphenicol.

Compressed tablets containing approximately 8 units of fibrinolysin and 6,000 units of desoxyribonuclease per tablet but no chloramphenicol can be prepared by the omission of chloramphenicol in the foregoing procedure.

EXAMPLE 5 Preparation of medicated film containing fibrinolysin and desoxyribonuclease A methyl cellulose product (15 cps.) having about 28-30% methoxy groups and about 7-12% hydroxypropoxy groups, and characterized by being soluble in both water and chloroform, is used in this preparation. The methyl cellulose product (7 g.) is dissolved in ml. of chloroform by rapid stirring. A mixture of fibrinolysin and desoxyribonuclease containing 123 units of fibrinolysinand 105,300 units of desoxyribonuclease is ground in a mortar with 7 m1. of propylene glycol and added to the methyl cellulose solution. The mixture is poured into a glass tray having an area of 936 square centimeters and the chloroform is evaporated at room temperature. The film which remains is cut into squares having an area of 64 square centimeters, suitable for vaginal administration.

EXAMPLE 6' Assay of fibrinolysin component Materials used in this assay include the following:

A standardized bovine fibrinolysin, the potency of which has been determined by repeated measurement of the amount required to lyse a standard 0.3% fibrin clot in 120 seconds at pH 7.2 and 45 C. in an isotonic saline system buffered with imidazole.

Standard bovine fibrinogen prepared according to the method of Archives of Biochemistry 13,231 (1947). The fibrinogen is stored in a lyophilized form containing imidazole in ml. vials containing sufficient material to perform one or two assays. To use the fibrinogen in the assay, 0.85% saline is added to prepare a solution corresponding to 0.6% clottable fibrinogen with imidazole to buffer at pH 7.2-7.4. The determination of clottable fibrinogen can be'carried out according to the procedure described by Johnson and Seegers in Tocantins, The Coagulation of Blood-Methods of Study, Grune & Stratton, New York, 1955, page 159-160. In order to prepare the standard fibrinogen solution for assay, sulficient 0.85% saline to give a solution corresponding to 0.6% clottable fibrinogen is added to a vial and solution is allowed to take place at room temperature. The solution isheld at room temperature because fibrinogen precipitates when it is chilled. Insoluble particles are removed by filtration through paper.

Thrombin (bovine origin) is freshly prepared in 0.85% saline (buffered to a pH of 7.2 with imidazole) in a concentration of 50 units per ml. and held in an ice bath.

In performing the assay, 0.3 ml. of the thrombin solution is delivered into each of a series of 3 to 5 10 x 75 mm. test tubes. In order to check the potency of a standard fibrinolysin solution prepared to contain approximately 5 units per ml., 0.2 ml. of the standard fibrinolysin solution is added to each of the tubes con- .taining thrombin which are then warmed in a 45 C. water bath for a few seconds. To each of the tubes is then added 0.5 ml. of the standard fibrinogen solution with tilting of the tubes to mix the reagents. A stop watch is started simultaneously. The tubes are placed in a water bath at 45 C., and a firm clot forms in each tube within 515 seconds. An open-ended capillary tube is inserted to the bottom of the clot and the time required for the liquid level in the capillary tube to rise to the top level of the clot in the test tube is recorded. For the standard fibrinolysin, this is found to average about 120 seconds. A unit of fibrinolysin is defined as that amount of fibrinolysin which will cause the end point to be reached in 120 seconds under the standard test conditions, that is, the amount of fibrinolysin which will dissolve 1 ml. of a 0.3% fibrin clot in 120 seconds at pH 7.2 and 45 C. in an isotonic saline system buffered with imidazole.

In order to determine the potency of an unknown fibrinolysin preparation, one or more trial dilutions are prepared and 0.2 ml. of each of these solutions is subjected to the assay to determine the lytic time. On the basis of these preliminary trials, two dilutions are then selected, one chosen to give a lytic time slightly less than and one slightly more than that obtained with one unit of the standard fibrinolysin. The potency of the unknown fibrinolysin preparation is then calculated by interpolation. For example, assume that 40 mg. of a powdered preparation containing fibrinolysin is dissolved in 1 ml. of 0.85% saline. A high dilution is prepared by diluting an aliquot of this solution with 80% of its volume of saline and a low dilution is prepared by dissolving an aliquot of this solution with 20% of its volume of saline. Assume that the lytic time of the high dilution is found to be 130 seconds and the" lytic time of the low dilution is found to be 100 seconds. The potency is calculated according to the formula: [(A B)+C] 5=units per ml., where A=(Factor relating the volume upon high dilution to the undiluted volume) minus (the factor relating the volume upon low dilution to the undiluted volume) B=(Standard lytic time)(lytic time of low dilution) (Lytic time of high dilution)(lytic time of low dilution) C=Factor relating the volume upon low dilution to the undiluted volume 5=Factor to convert to units per ml. since 0.2 m1. is used in the test A=1.81.2=0.6 B=l20100/130100=% C=1.2

Consequently, the original solution prepared by dissolving 40 mg. of powder fibrinolysin preparation in 1 ml. of saline contained 8 units per ml. and the powdered fibrinolysin preparation itself contained 0.2 unit per mg.

Where the potency of the unknown solution is too low to give lytic times of seconds or less, it is not possible to bracket the lytic times obtained with the standard fibrinolysin solution. For such cases, a table of lytic times corresponding to fibrinolysin units per ml. can be prepared from a curve in which there is plotted lytic times of various dilutions against fibrinolysin units per 0.2 ml. used in the test, assuming a lytic time of 120 seconds always equals 1 unit by definition.

EXAMPLE 7 Assay of desoxyribonuclease component Materials used in this assay include the following:

A Veronal buffer prepared by adding 13.0 ml. of 0.50 N sodium hydroxide to a suspension of 4.60 g. of Veronal in 900 ml. of water, warming to dissolve, cooling, adjusting the pH to about 7.50 with dilute sodium hydroxide, and diluting to 1000 ml. with water.

A Veronal-magnesium sulfate solution, prepared by dissolving 0.616 g. of magnesium sulfate heptahydrate in 500 ml. of the Veronal buffer.

A Veronal-magnesium sulfate-gelatin solution, prepared by dissolving 0.625 g. of gelatin in 500 ml. of the Veronalmagnesium sulfate solution.

Highly polymerized sodium desoxyribonucleate from which solutions falling within the required range of viscosities are prepared. The sodium desoxyribonucleate is suspended in Veronal-magnesium sulfate solution at a level of about 5 mg./ml. After it has stood for 18 hours at 0-5" C., the mixture is homogenized thoroughly in a Potter-Elvejhem type homogenizer, diluted with an equal volume of Veronal-magnesium sulfate solution, again homogenized, and allowed to stand at 0 C. for 30 minutes. The relative viscosity is then determined by diluting 2.3 ml. of this solution to 2.5 ml. with Veronal-magnesium sulfate-gelatin solution and measuring the flow time at 30 C. in a viscometer in which 2.5 ml. of water has a flow time of no more than 20 seconds and no less than 10 seconds. Gradual dilutions of the original solution are then made, by trial and error, repeating after each dilution, the homogenizing, standing at 0 C. and determination of relative viscosity. Each relative viscosity measurement is made with a 2.3 ml. aliquot of the particular dilution of sodium desoxyribonucleate, to which has been added 0.2 ml. of Veronal-magnesium sulfategelatin solution. Dilutions are continued until a solution is obtained which has a relative viscosity of no more than 4.4 and no less than 4.2 when 2.3 ml. is diluted to 2.5 ml. with Veronal-magnesium sulfate-gelatin solution.

For performing the assay, viscometers are set up in a water bath at 30 C. Various dilutions of the desoxyribonuclease prepartion of unknown potency in Veronalmagnesium sulfate-gelatin solution, and 2.3 m1. aliquots of the sodium desoxyribonucleate solution are kept in test tubes in an ice bath.

A 0.2 ml. portion of a desoxyribonuclease solution of one of the selected dilutions is added to a 2.3 ml. aliquot of the sodium desoxyn'bonucleate solution of required viscosity and a stop watch is simultaneously started. The mixture is rapidly mixed by inversion and poured into a viscometer. Viscometric measurements are started immediately and made continuously for about 15 minutes until enough points are obtained to establish a satisfactory curve by plotting relative viscosity against elapsed time, the time of measurement in the case of each point being taken as midway between the beginning and the end of the measurement.

A unit of desoxyribonuclease is defined as that amount of desoxyribonuclease which causes a fall in relative viscosity of 1.0 under the standard assay condition, with the restriction that this fall in relative viscosity of 1.0 be between relative viscosity:4.0 and relative viscosity:3.0 during a -minute period. Therefore, it is preferable that dilutions of desoxyribonuclease be chosen such that during the 10-minute period following the time at which relative viscosity:4.0, the fall in relative viscosity, for one dilution of the enzyme is between 0.4 and 1.0, for another dilution of the enzyme close to 1.0, and for a third dilution of the enzyme between 1.0 and 2.5. For each of the selected dilutions of desoxyribonuclease a curve is constructed by plotting relative viscosity against elapsed time. From each curve, the change'in relative viscosity for the 10-minute period following the time at which relative viscosity:4.0 is obtained; this change in relative viscosity is characteristic of the enzyme dilution on which the curve is based. The three values obtained for change in relative viscosity (one from, each curve) are then plotted against the quantities of desoxyribonuclease preparation in 0.2 ml. of the corresponding dilutions and from the resulting curve the amount of desoxyribonuclease preparation required to cause the defined unit fall in relative viscosity is obtained by interpolation.

We claim:

1. A therapeutic composition for enzymatic debridement comprising, in combination, fibrinolysin and desoxy-' ribonuclease, said fibrinolysin and said desoxyribonuclease being present in a ratio of 1 unit of fibrinolysin to 20-5000 units of desoxyribonuclease.

2. A method for facilitating the healing of lesions by nuclease.

References Cited by the Examiner UNITED STATES PATENTS 2,624,691 1/ 53 Loomis 167-73 X 2,801,956 8/57 Baumgarten et al 167-73 X 2,834,710 5/58 Baumgarten et al. l67-73 X- OTHER REFERENCES Armstrong et al.: Lancet, volume 259, pages 739-42, December 9,1950. 7

' Bird: Surgery, 42 (1), pages 249-55, July 1957.

Fullgrabe: Annals New York Acad. Sci., 68(1), pages 192-5, August 30, 1957.

J.A.M.A., 158 (16), page Lesser: Drug and Cosm. Ind., 250-4, August 1952.

Lichtman: Annals New York Acad. Sci., 196-200, August 30, 1957.

Modern Drugs, January 1954, page 693.

Neff: Am. J. Pharmacy, 131 (1), pages 7-13, January 1959.

Raker: Ann. New York Acad. Sci., 68 (1), 50, August 30, 1957.

Robinson: Lancet, 2 (7051), 18, 1958.

1433, August 2, 1955.

71 (2), pages 178, 9 and 68 1) pages pages 144- pages 819-21, October FRANK CACCIAPAGLIA, 111., Primary Examiner.

, LEWIS GOTTS, Examiner. 

1. A THERAPEUTIC COMPOSITION FOR ENZYMATIC DEBRIDEMENT COMPRISING, IN COMBINATION, FIBRINOLYSIN AND DESOXYRIBONUCLEASE, SAID FIBRINOLYSIN AND SAID DESOXYRIBONUCLEASE BEING PRESENT IN A RATIO OF 1 UNIT OF FIBRINOLYSIN TO 20-5000 UNITS OF DESOXYRIBONUCLEASE. 